RIBONUCLEASE SUPERFAMILY--STRUCTURE-FUNCTION STUDIES

Project: Research project

Project Details

Description

This project is concerned with obtaining detailed structure-function
correlations for members of the ribonuclease superfamily. This family
of proteins includes digestive enzymes such bovine pancreatic
ribonuclease (RNase A), toxins, such as human eosinophil derived
neurotoxin, growth factors such as human angiogenin, and P-30, a protein
with significant toxicity against human tumor cell lines. The sequence
homology and ribonucleolytic activity of all these proteins, suggests
that they share a common catalytic mechanism and tertiary structure. The
vast amount of structural and biochemical information available for RNase
A provides a unique opportunity to examine the basis for the biological
activities displayed by members of the ribonuclease superfamily. RNase
A will be systematically mutated towards the primary sequence of these
homologs, and the hybrid proteins will be examined by X-ray
crystallography, kinetics and the appropriate bioassays.

In every case, the biological activities have been linked to the
ribonucleolytic activity. It is therefore crucial to understand the
detailed enzymatic mechanism(s) used by the various homologs. In
addition to unraveling the connection between enzymatic activity and
biological function, the production of hybrid proteins will allow for the
study of phenomena which are of fundamental importance to enzymology.
Specifically, members of the ribonuclease superfamily utilize the binding
energy of extended substrates to enhance catalytic efficiency by up to
four orders of magnitude. Furthermore, the rate of catalysis in the
superfamily is highly sensitive to the identity of amino acids which are
not directly involved in either the chemical transformation or substrate
binding: kcat/Km spans six orders of magnitude across the superfamily.
How enzymatic activity is effected by interactions distant from the site
of chemistry is one of the outstanding questions in enzymology, and is
of general importance.

The production of hybrid ribonucleases will allow for an examination of
important biological activities such as neurotoxicity, growth factor
activity and tumor cytotoxicity. In addition, these same studies will
address the fundamental question of 'long range distant effects' in
catalysis. Defining the structural and chemical determinants responsible
for these biological activities will be instrumental in understanding
their underlying mechanism and in providing a basis for therapeutic
development.
StatusFinished
Effective start/end date8/1/947/31/95

Funding

  • National Institute of General Medical Sciences

ASJC

  • Catalysis

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